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Figure 1: Chromatography for phospholipids observed in a standard bioanalytical method, uising 4 different sample preparation approaches 5µ C18 50 x 4.6mm HPLC column, flow rate of 1mL/min Mobile phase A was 10mM ammonium acetate (aq) Mobile phase B was Acetonitrile 0 30%, 2.25 mins to 80%, step to 100% for 2.25 mins, then to 30% reequilibration for 2 mins
Figure 2: Demonstrating how phospholipids can elute from a HPLC column. 1st injection is a protein crash sample of rat plasma, all subsequent injections are water.
may not be correct, in particular if only a few of the components that are injected are being monitored. This is the scenario in many bioanalytical assays where the mass spectrometer is used to add further resolving power to the chromatographic separation by only monitoring for the analytes of interest. Unlike when using a less discriminative detector, matrix components that elute from the column will typically not be monitored, and thus it may not be feasible to determine if the peaks elute over one gradient cycle or require more gradient cycles to fully elute the matrix components form the column. This will result in variable amounts of matrix components being eluted dependant on the injection number within a particular batch.
Figures 1 and 2 looks at the elution of a series of phospholipids. Figure 1 shows the chromatography and responses obtained using different sample preparation techniques. It can be seen that from Figure 1 the peak shape is poor, and that a reasonable amount of retention time space is covered by just three matrix components. Altering the sample preparation also has a significant impact in the amount of phospholipids that are left on the column, with a protein precipitation approach providing the worst approach to removing this particular matrix component. Figure 2 investigates how effectively the
Figure 3
phospholipids are eluted from a column. The first injection is from a protein precipitated sample and it can be seen that there is a high amount of the lower molecular mass (and lower hydrophobicities) phospholipids. All subsequent injections are of water, and so any peaks that are eluting are due to system carryover. In this case it is very evident that the chromatography has not been optimised for the matrix components and this results in the phospholipids being eluted from the column over many injections. In a standard batch the amount of matrix components in different samples can vary substantially, particularly if a less effective sample preparation technique is employed. This results in potentially a very complex situation for the amount of matrix components that are eluting into the detector, as it will be dependent not only on the on the original sample, but also on the history of the column.
The issue with the variable matrix components is that each matrix component has the potential to interfere with the ionisation process, due to the nature of how an ESI source works. Figure 3 shows a schematic of an ESI source. An ESI source is designed such that the
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